Sudha et al., IJPSR, 2010; Vol. 1 (11): 107-111 ISSN: 0975-8232
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IJPSR (2010), Vol. 1, Issue 11 (Research Article)
Received on 22 August, 2010; received in revised form 17 October, 2010; accepted 26 October, 2010 VALIDATED HPTLC METHOD FOR SIMULTANEOUS DETERMINATION OF LAMIVUDINE AND ABACAVIR SULPHATE IN TABLET DOSAGE FORM
T. Sudha*1, V. R. Ravikumar 2 and P. V. Hemalatha 2
Department of Pharmaceutical Analysis 1, Department of Pharmacognosy 2, The Erode College of Pharmacy& Research Institute, Erode, Tamil Nadu, India
ABSTRACT
A simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and validated for the simultaneous estimation of Lamivudine and Abacavir sulphate in combined dosage forms. The stationary phase was precoated silica gel 60F254. The
mobile phase used was a mixture of (Acetone: chloroform: methanol 4: 4: 2 v/v/v). The detection of spot was carried out at 265nm. The method was validated in terms of linearity, accuracy, precision and specificity. The calibration curve was found to be linear between 500 to 3000 ng with regression coefficient of 0.9998. The proposed method can be successfully used to determine the drug content of marketed formulation.
Keywords:
Abacavir sulphate, Lamivudine, Simultaneous Estimation,
HPTLC, Validation
Correspondence to Author:
T. Sudha
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INTRODUCTION: The combination of Lamivudine (LAM) and Abacavir (ABA) has recently been introduced in the market. Chemically Lamivudine
1
is (Lam) 4 amino-1- [2R, 5S) - 2- (hydroxyl methyl) - 1, 3- oxathiolan- 5yl] pyrimidine-2-one. Lamivudine is a nucleoside reverse transcriptase inhibitor (NRTI) with an activity against human immunodeficiency virus type (1) (HIV) 1 and hepatitis B. Abacavir 2 is (Aba) [1R] - 4- (2 amino-6-cyclopyridine) purine- 9- yl) -1- cyclopent- 2 enyl] methanol. It is a nucleoside reverse transcriptase inhibitor (NRTI) with an activity against human immune deficiency virus Type (1) (HIV1).
LAMIVUDINE
ABACAVIR SULPHATE
The drugs are prescribed invidually, as well as multicomponent dosage forms available in the market. A number of methods have been
published for the estimation of above said analytes.
Spectrophotometric estimation of Abacavir sulphate 3
Spectrophotometric estimation of Lamivudine
4
Lamivudine in human plasma by RP-HPLC 5
Titrimetric and spectrophotometric estimation of Lamivudine 6
A method was reported for simultaneous analysis of Abacavir and Lamivudine in human plasma by LC/MS/MS 7. Determination of Abacavir, Lamivudine, Zidovudine in pharmaceutical tablets human serum and in drug dissolution studies by HPLC 8 was also reported in the literature. Literature survey reveals that so, far no HPTLC method has been reported for the simultaneous estimation of ABA and Lam formulation. In the present HPTLC studies investigation, an attempt have been made to develop a rapid, accurate, precise and cost effective HPTLC method for simultaneous estimation of LAM and ABA in combined dosage form.
MATERIALS AND METHODS: LAM and ABA
standards were procured as a gift samples from Hetero labs Hyderabad. Silica gel 60F254TLC
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precoated silica gel 60F254 aluminum sheets
(4x10cm) as stationary phase, Acetone: chloroform: methanol (4: 4: 2 v/v/v) as mobile phase, chamber and plate saturation time of 30 min, migration distance was 90 mm, wavelength was selected by scanning standard solution of both drugs over 200 nm to 400 nm. LAM showed maximum absorbance at 366 nm and ABA at 254 nm. Both components showed reasonably good response at 265 nm. Therefore photometric measurements were performed at 265 nm absorption mode with cagmag TLC scanner 3 using win CATS soft ware. Stock solutions of LAM and ABA were prepared by separately dissolving 25 mg of Aba and 25 mg of Lam in 25 ml methanol. Further dilution was made by diluting 2.5 ml with mobile phase to obtain 50 µg/ml solutions. Working stock solution were prepared by diluting the stock solution with mobile phase to obtain final concentration of 5, 10, 15, 20, 25 and 30 µg/ml of both drugs (LAM and ABA ) were applied separately on the TLC plate.
TLC plate was dried, developed and analyzed photometrically as described earlier. The calibration graph was plotted using peak area against concentration. The procedure was repeated for three times to determine the LOD and LOQ. The marketed formulation Abamune-L contains (300mg of LAM and 600mg of ABA). Twenty tablets were weighed accurately, finely powered and mixed. The average mass per tablet was determined. The powder tablet equivalents to 25 mg of each were accurately weighed and added a minimum quantity of methanol to dissolve the substance the total volume as brought to 25 ml with more methanol (1000 µg/ml) in a volumetric flask. The solutions were sonicated for 10 minutes and then filtered through whatmann filter paper No 41 to separate out the insoluble excipients. Collect the filtrate after rejecting the first portion of the filtrate.
From the clear solution, further dilutions were made by diluting 2.5ml to 50ml with mobile phase to obtain 50 µg/ml further dilution was made by diluting 3 ml to 10 ml with mobile phase to obtain 15 µg/ml. 15 micro liters of sample solutions were spotted on to the TLC plate and developed. The analysis was repeated for six times. The content of the drug was calculated from the peak areas recorded. The accuracy of the method was confirmed by recovery studies. The recovery was performed at three different concentrations (20%, 40%, 60%) were added to fixed amount of pre analyzed sample and the amount of each of the drug were determined by the proposed method. Further the precision of the developed method was confirmed by interday and intraday analysis.
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analyte and area under the peak area. The proposed method was validated as per ICH guidelines 10, 11.
FIG.1: A TYPICAL HPTLC CHROMATOGRAM OF LAM AND ABA
[image:4.612.310.563.86.243.2]Limit of Quantification was found to be 0.0254 and 0.0105 µg/ml for LAM and ABA respectively. Limit of detection was found to be 0.0083 and 0.0034 µg/ml for LAM and ABA respectively (Table 1). Precision is the degree of reproducibility or repeatability of the method under normal operating condition. The method passed the test for repeatability as determined by % RSD 12 of the peak area of six replicate. The % concentration of LAM and ABA were found to be 100.01 ± 0.4300 and 99.50 ± 0.5911 respectively.
TABLE-1: METHOD VALIDATION PARAMETERS
Parameters Values
Lamivudine Abacavir
Linearity range 5-30µg/ml 5-30µg/ml
Correlation
coefficient 0.9996 0.9998
Regression
equation Y=1993.37X+571.05 Y= 3965.88X+279.30
Slope 1993.37 3965.88
Intercept 571.02 279.30
Limit of
detection 0.0083 0.0034
Limit of
Quantification 0.0254 0.0105
The low % RSD value indicated that the method has good precision. The results of the analysis are shown in Table 2. Further the precision of the developed method is confirmed by interday and intraday analysis. The result show good agreement with the label claim of the formulation. To evaluate the accuracy of the method, known amount of pure drug was added to the previously analyzed solution containing pharmaceutical formulation and the mixture was analyzed by the proposed method and the recoveries were calculated.
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TABLE 2: ASSAY OF LAM AND ABA
Formulation Drugs Label claim Amount found %purity Average SD %RSD
Abamune-L
Lamivudine 300mg
298.642 99.54
100.01 0.4300 0.4299
301.604 100.53 300.114 100.03 299.190 99.73 299.164 99.72 301.604 100.53
Abacavir 600mg
599.190 99.86
99.50 0.5911 0.5940
603.285 100.54 594.190 99.03 595.266 99.21 596.190 99.36 594.285 99.04 TABLE-3: RECOVERY STUDIES OF LAM AND ABA
Label claim Amount Added Amount
Recovered % Recovery Average SD %RSD
Lam 300mg
3.00 6.00 9.00
3.01 6.01 8.99
100.33 100.16 99.88
100.12 0.2272 0.2269
Aba600mg 6.0
12.0 18.0
5.99 12.05 17.99
99.83 100.41
99.94
100.06 0.3080 0.3078
CONCULSION: The HPTLC method developed for LAM and ABA shows good precision and accuracy. The low % RSD value in the recovery studies indicates that there is no excepients used in the formulation. Hence, it is concluded that the developed method is simple, precise, accurate and rapid for the analysis of LAM and ABA in pure and tablet dosage form. Then the developed method can be adopted for the routine analysis of LAM and ABA in bulk and in tablet dosage form.
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